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Oligonucleoside Phosphorothioates

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Protocols for Oligonucleotides and Analogs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 20))

Abstract

There are three constitutionally isomeric forms of internucleoside phosphorothioate linkages. Replacement of either the 3′- or 5′-oxygen in a phosphodiester linkage, 1, results in the respective thiolo structures 2 or 3, whereas substitution of a nonbridging oxygen with sulfur gives a thiono structure 4. Oligonucleoside thiolo phosphorothioates 2 and 3, which have an achiral phosphorus moiety and are electronically similar to 1, are currently not synthesized by automated methods (1,2) as easily as their thiono counterpart (3), 4. Thiono form 4 also differs from 2 and 3 by having a chiral phosphorus (R p , S p ) center and a negative charge localized on sulfur 5 (4,5).

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References

  1. Cosstick, R. and Vyle, J. S. (1989) Solid phase synthesis of oligonucleotides containing 3−-thiothymidine. Tetrahedron Lett. 30, 4693–4696.

    Article  CAS  Google Scholar 

  2. Mag, M., Lüking, S., and Engels, J. W. (1991) Synthesis and selective cleavage of an oligodeoxynucleotide containing a bridged internucleotide 5−-phosphorothioate linkage. Nucl. Acids Res. 19, 1437–1441.

    Article  CAS  Google Scholar 

  3. Zon, G. and Stec, W. J. (1991) Phosphorothioate oligonucleotides, in Oligonucleotides and Their Analogues (Eckstein, F., ed.) IRL Press, London, pp. 87–108.

    Google Scholar 

  4. Liang, C. and Allen, L. C. (1987) Sulfur does not form double bonds in phosphorothioate anions. J. Am. Chem. Soc. 109, 6449–6543.

    Article  CAS  Google Scholar 

  5. Baraniak, J. and Frey, P. A. (1988) Effect of ion pairing on bond order and charge localization in alkyl phosphorothioates. J. Am. Chem. Soc. 110, 4059–4060.

    Article  CAS  Google Scholar 

  6. Eckstein, F. (1967) A dinucleoside phosphorothioate. Tetrahedron Lett. 1157–1160.

    Google Scholar 

  7. Eckstein, F. (1967) Diuridin-3′, 5′-thiophosphat. Tetrahedron Lett. 3495–3499.

    Google Scholar 

  8. Matzura, H. and Eckstein, F. (1968) A polyribonucleotide containing alternating → P=O and → P=S linkages. Eur. J. Biochem. 3, 448–452.

    Article  CAS  Google Scholar 

  9. Eckstein, F. and Gindl, H. (1970) Polyribonucleotides containing a phosphorothioate backbone. Eur. J. Biochem. 13, 558–564.

    Article  CAS  Google Scholar 

  10. Eckstein, F. (1985) Nucleoside phosphorothioates. Ann. Rev. Biochem. 54, 367–402.

    Article  CAS  Google Scholar 

  11. Gish, G. and Eckstein, F. (1988) DNA and RNA sequence determination based on phosphorothioate chemistry. Science 240, 1520–1521.

    Article  CAS  Google Scholar 

  12. Eckstein, F. and Gish, G. (1989) Phosphorothioates in molecular biology. Trends in Biochem. Sci. 14, 97–100.

    Article  CAS  Google Scholar 

  13. Herschlag, D., Piccirilli, J. A., and Cech, T. R. (1991) Ribozyme-catalyzed and nonenzymatic reactions of phosphate diesters: rate effects upon substitution of sulfur for a nonbridging phosphoryl oxygen atom. Biochemistry 30, 4844–4854.

    Article  CAS  Google Scholar 

  14. Matsukura, M., Shinozuka, K., Zon, G., Mitsuya, H., Reitz, M., Cohen, J. S., and Broder, S. (1987) Phosphorothioate analogs of oligodeoxynucleotides: inhibitors of replication and cytopathic effects of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 84, 7706–7710.

    Article  CAS  Google Scholar 

  15. Matsukura, M., Zon, G., Shinozuka, K., Stein, C. A., Mitsuya, H., and Broder, S. (1988) Synthesis of phosphorothioate analogues of oligodeoxyribonucleo-tides and their antiviral activity against human immunodeficiency virus (HIV). Gene 72, 343–347.

    Article  CAS  Google Scholar 

  16. Agrawal, S., Goodchild, J., Civeira, M. P., Thornton, A. H., and Zamecnik, P. C. (1988) Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 85, 7079–7083.

    Article  CAS  Google Scholar 

  17. Agrawal, S., Ikeuchi, T., Sun, D., Sarin, P. S., Konopk, A., and Zamecnik, P. C. (1989) Inhibition of human immunodeficiency virus in early infected and chronically infected cells by antisense oligodeoxynucleotides and their phosphorothioate analogues. Proc. Natl. Acad. Sci. USA 86, 7790–7794.

    Article  CAS  Google Scholar 

  18. Letsinger, R. L., Zhang, G., Sun, D. K., Ikeuchi, T., and Sarin, P. S. (1989) Cholesteryl-conjugated oligonucleotides: Synthesis, properties, and activity as inhibitors of replication of human immunodeficiency virus in cell culture. Proc. Natl. Acad. Sci. USA 86, 6553–6556.

    Article  CAS  Google Scholar 

  19. Stein, C. A., Matsukura, M., Subasinghe, C., Broder, S., and Cohen, J. S. (1989) Phosphorothioate oligodeoxynucleotides are potent sequence nonspecific inhibitors of de novo infection by HIV. Aids Res. Human Retroviruses 5, 639–646.

    Article  CAS  Google Scholar 

  20. Matsukura, M., Zon, G., Shinozuka, K., Robert-Guroff, M., Shimada, T., Stein, C. A., Mitsuya, H., Wong-Staal, F., Cohen, J. S., and Broder, S. (1989) Regulation of viral expression of human immunodeficiency virus in vitro by an antisense phosphorothioate oligodeoxynucleotide against rev (art/trs) in chronically infected cells. Proc. Natl. Acad. Sci. USA 86, 4244–4248.

    Article  CAS  Google Scholar 

  21. Shibahara, S., Mukai, S., Morisawa, H., Nakashima, H., Kobayashi, S., and Yamamoto, N. (1989) Inhibition of human immunodeficiency virus (HIV-1) replication by synthetic oligo-RNA derivatives. Nucl. Acids Res. 17, 239–252.

    Article  CAS  Google Scholar 

  22. Majumdar, C, Stein, C. A., Cohen, J. S., and Broder, S. (1989) Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide. Biochemistry 28, 1340–1346.

    Article  CAS  Google Scholar 

  23. Iyer, R. P., Uznanski, B., Boal, J., Storm, C, Egan, W., Matsukura, M., Broder, S., Zon, G., Wilk, A., Koziolkewicz, M., and Stec, W. J. (1990) Abasic oligode-oxyribonucleoside phosphorothioates: Synthesis and evaluation as anti-HIV-1 agents. Nucl. Acids Res. 18, 2855.

    Article  CAS  Google Scholar 

  24. Leiter, J. M. E., Agrawal, S., Palese, P., and Zamecnik, P. C. (1990) Inhibition of influenza virus replication by phosphorothioate oligodeoxynucleotides. Proc. Natl. Acad. Sci. USA 87, 3430–3434.

    Article  CAS  Google Scholar 

  25. Goodarzi, G., Gross, S. C, Tewari, A., and Watabe, K. (1990) Antisense oligodeoxyribonucleotides inhibit the expression of the gene for hepatitis B virus surface antigen. J. Gen. Virol. 71, 3021–3025.

    Article  CAS  Google Scholar 

  26. Ceruzzi, M. and Draper, K. (1989) The intracellular and extracellular fate of oligodeoxyribonucleotides in tissue culture systems. Nucleosides & Nucleotides 8, 815–818.

    Article  Google Scholar 

  27. Gao, W., Stein, C. A., Cohen, J. S., Dutschman, G.E., and Cheng, Y. C. (1989) Effect of phosphorothioate homo-oligodeoxynucleotides on herpes simplex virus type 2-induced DNA polymerase. J. Biol. Chem. 264, 11,521–11,526.

    CAS  Google Scholar 

  28. Gao, W. Y., Hanes, R. N., Vazquez-Padua, M. A., Stein, C. A., Cohen, J. S., and Cheng, Y. C. (1990) Inhibition of herpes simplex virus type 2 growth by phosphorothioate oligodeoxynucleotides. Antimicrobial Agents and Chemotherapy 34, 808–812.

    CAS  Google Scholar 

  29. Shea, R. G., Marsters, J. C, and Bischofberger, N. (1990) Synthesis, hybridization properties and antiviral activity of lipid-oligodeoxynucleotide conjugates. Nucl. Acids Res. 18, 3777–3783.

    Article  CAS  Google Scholar 

  30. Storey, A., Oates, D., Banks, L., Crawford, L., and Crook, T. (1991) Antisense phosphorothioate oligonucleotides have both specific and nonspecific effects on cells containing human papillomavirus type 16. Nucl. Acids Res. 19, 4109–4114.

    Article  CAS  Google Scholar 

  31. Manson, J., Brown, T., and Duff, G. (1990) Modulation of interleukin iβ gene expression using antisense phosphorothioate oligonucleotides. Lymphokine Res. 9, 35–42.

    CAS  Google Scholar 

  32. Cazenave, C., Stein, C. A., Loreau, N., Thuong, N. T., Neckers, L. M., Subasinghe, C, Helene, C, Cohen, J. S., and Toulmé, J. J. (1989) Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides. Nucl. Acids Res. 17, 4255–4273.

    Article  CAS  Google Scholar 

  33. Woolf, T. M., Jennings, C. G. B., Rebagliati, M., and Melton, D. A. (1990) The stability, toxicity and effectiveness of unmodified and phosphorothioate antisense oligodeoxynucleotides in Xenopus oocytes and embryos. Nucl. Acids Res. 18, 1763–1769.

    Article  CAS  Google Scholar 

  34. Agrawal, S., Mayrand, S. H., Zamecnik, P. C, and Pederson, T. (1990) Site-specific excision from RNA by RNase H and mixed-phosphate-backbone oligodeoxynucleotides. Proc. Natl. Acad. Sci. USA 87, 1401–1405.

    Article  CAS  Google Scholar 

  35. Furdon, P. J., Dominski, Z., and Kole, R. (1989) RNase II cleavage of RNA hybridized to oligonucleotides containing methylphosphonate, phosphorothioate and phosphodiester bonds. Nucl. Acids Res. 17, 9193–9204.

    Article  CAS  Google Scholar 

  36. Baker, C., Holland, D., Edge, M., and Colman, A. (1990) Effects of oligo sequence and chemistry on the efficiency of oligodeoxyribonucleotide-mediated mRNA cleavage. Nucl. Acids Res. 18, 3537–3543.

    Article  CAS  Google Scholar 

  37. Dagle, J. M., Walder, J. A., and Weeks, D. L. (1990) Targeted degradation of mRNA in Xenopus oocytes and embryos directed by modified oligonucleotides: Studies of An2 and cyclin in embryogenesis. Nucl. Acids Res. 18, 4751–4757.

    Article  CAS  Google Scholar 

  38. Kumble, K. D., Iversen, P. L., and Vishwanatha, J. K. (1992) The role of primer recognition in DNA replication: inhibition of cellular proliferation by antisense oligodeoxyribonucleotides. J. Cell Sci. 101, 35–41.

    CAS  Google Scholar 

  39. Marcus-Sekura, C. J., Woerner, A.M., Shinozuka, K., Zon, G., and Quinnan, G. V. (1987) Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages. Nucl. Acids Res. 15, 5749–5763.

    Article  CAS  Google Scholar 

  40. Young, S. and Wagner, R. W. (1991) Hybridization and dissociation rates of phosphodiester or modified oligodeoxynucleotides with RNA at near-physiological conditions. Nucl. Acids Res. 19, 2463–2474.

    Article  CAS  Google Scholar 

  41. Chang, E. H., Yu, Z., Shinozuka, K., Zon, G., Wilson, W. D., and Strekowska, A. (1989) Comparative inhibition of ras p21 protein synthesis with phosphorus-modified antisense oligonucleotides. Anti-Cancer Drug Design 4, 221–232.

    CAS  Google Scholar 

  42. Efcavitch, J. W. (1988) Automated system for the optimized chemical synthesis of oligodeoxyribonucleotides, in Macromolecular Sequencing and Synthesis Selected Methods and Applications (Schlesinger, D. H., ed.) Liss, New York, pp. 221–234.

    Google Scholar 

  43. Vu, H. and Hirschbein, B. L. (1991) Internucleotide phosphite sulfurization with tetraethylthiuram disulfide phosphorothioate oligonucleotide synthesis via phosphoramidite chemistry. Tetrahedron Lett. 32, 3005–3008.

    Article  CAS  Google Scholar 

  44. Zon, G. (1990) Purification of synthetic oligodeoxyribonucleotides, in High Performance Liquid Chromatography in Biotechnology (Hancock, W. S., ed.) Wiley, New York, pp. 301–397.

    Google Scholar 

  45. Stein, C. A., Iversen, P. L., Subasinghe, C., Cohen, J. S., Stec, W. J., and Zon, G. (1990) Preparation of 35S-labeled polyphosphorothioate oligodeoxyribonucleotides by use of hydrogen phosphonate chemistry. Anal. Biochem. 188, 11–16.

    Article  CAS  Google Scholar 

  46. Eadie, J. S., McBride, L. J., Efcavitch, W., Hoff, L. B., and Cathcart, R. (1987) High-performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition. Anal. Biochem. 165, 442–447.

    Article  CAS  Google Scholar 

  47. Stec, W. J., Zon, G., Egan, W., and Stec, B. (1984) Automated solid-phase synthesis, separation, and stereochemistry of phosphorothioate analogues of oligodeoxyribonucleotides. J. Am. Chem. Soc. 106, 6077–6079.

    Article  CAS  Google Scholar 

  48. Ott, J. and Eckstein, F. (1987) Protection of oligonucleotide primers against degradation by DNA polymerase I. Biochemistry 26, 8237.

    Article  CAS  Google Scholar 

  49. Froehler, B. C. (1986) Deoxynucleoside H-phosphonate diester intermediates in the synthesis of internucleotide phosphate analogues, Tetrahedron Lett. 27, 5575–5578.

    Article  CAS  Google Scholar 

  50. Andrus, A., Efcavitch, J. W., McBride, L. J., and Giusti, B. (1988) Novel activating and capping reagents for improved hydrogen-phosphonate DNA synthesis. Tetrahedron Lett. 29, 861–864.

    Article  CAS  Google Scholar 

  51. Iyer, R. P., Phillips, L. R., Egan, W., Regan, J. B., and Beaucage, S. L. (1990) The automated synthesis of sulfur-containing oligodeoxyribonucleotides using 3H-l,2-benzodithiol-3-one 1,1-dioxide as a sulfur-transfer reagent. J. Org. Chem. 55, 4693–4699.

    Article  CAS  Google Scholar 

  52. Kamer, P. C. J., Roeien, H.C. P. F., van den Elst, H., van der Marel, G. A., and van Boom, J. H. (1989) An efficient approach toward the synthesis of phosphorothioate diesters via the Schonberg reaction. Tetrahedron Lett. 30, 6757–6760.

    Article  CAS  Google Scholar 

  53. Stawinski, J. and Thelin, M. (1991) Nucleoside H-phosphonates. 13. Studies on 3H-l,2-benzodithiol-3-one derivatives as sulfurizing reagents for H-phosphonate and H-phosphonothioate diesters. J. Org. Chem. 56, 5169–5175.

    Article  CAS  Google Scholar 

  54. Slim, G. and Gait, M. J. (1991) Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes. Nucl. Acids Res. 19, 1183–1188.

    Article  CAS  Google Scholar 

  55. Morvan, F., Rayner, B., and Imbach, J. L. (1990) Modified oligonucleotides: IV. Solid phase synthesis and preliminary evaluation of phosphorothioate RNA as potential antisense agents. Tetrahedron Lett. 31, 7149–7152.

    Article  CAS  Google Scholar 

  56. Zon, G. and Thompson, J.A. (1986) A review of high-performance liquid chromatography in nucleic acids research. BioChromatography 1, 22–32.

    CAS  Google Scholar 

  57. McBride, L. J., McCollum, C., Davidson, S., Efcavitch, J. W., Andrus, A., and Lombardi, S. J. (1988) A new, reliable cartridge for the rapid purification of synthetic DNA, BioTechniques 6, 362–367.

    Article  CAS  Google Scholar 

  58. Eadie, J. S. and Davidson, D. S. (1987) Guanine modification during chemical DNA synthesis. Nucl. Acids Res. 15, 8333–8349.

    Article  CAS  Google Scholar 

  59. Stec, W. J. and Zon, G. (1984) Synthesis, separation, and stereochemistry of diastereomeric oligodeoxyribonucleotides having a 5′-terminal internucleotide phosphorothioate linkage. Tetrahedron Lett. 25, 5275–5278.

    Article  CAS  Google Scholar 

  60. Hodges, R. R., Conway, N. E., and McLaughlin, L. W. (1989) “Post-assay” covalent labelling of phosphorothioate-containing nucleic acids with multiple fluorescent markers. Biochemistry 28, 261–267.

    Article  CAS  Google Scholar 

  61. Fidanza, J. A. and McLaughlin, L. W. (1989) Introduction of reporter groups at specific sites in DNA containing phosphorothioate diesters. J. Am. Chem. Soc. 111, 9117–9119.

    Article  CAS  Google Scholar 

  62. Agrawal, S. and Zamecnik, P. C. (1990) Site specific functionalization of oligonucleotides for attaching two different reporter groups. Nucl. Acids Res. 18, 5419–5423.

    Article  CAS  Google Scholar 

  63. Connell, C, Fung, S., Heiner, C, Bridgham, J., Chakerian, V., Heron, E., Jones, B., Menchen, S., Mordan, W., Raff, J., Recknor, M., Smith, L., Springer, J., Woo, S., and Hunkapiller, M. (1987) Automated DNA sequence analysis. BioTechniques 5, 342–347.

    CAS  Google Scholar 

  64. Chin, D. J., Green, G. A., Zon, G., Szoka, F.C., and Straubinger, R. M. (1990) Rapid nuclear accumulation of injected oligodeoxyribonucleotides. The New Biologist 2, 1091–1100.

    CAS  Google Scholar 

  65. Iversen, P. L., Zhu, S., Meyer, A., and Zon, G. (1992) Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells. Antisense Res. Dev. 2.

    Google Scholar 

  66. Marti, G., Egan, W., Noguchi, P., Zon, G., Matsukura, M., and Broder, S. (1992) Oligodeoxyribonucleotide phosphorothioate fluxes and localization in hematopoietic cells. Antisense Res. Dev. 2, 27–39.

    CAS  Google Scholar 

  67. Bergot, B. J. and Egan, W. (1992) Separation of synthetic phosphorothioate oligodeoxynucleotides from their oxygenated (phosphodiester) defect species by strong-anion exchange high-performance liquid chromatography. J. Chromatogr. 599, 34–42.

    Article  Google Scholar 

  68. Geiser, T. (1990) Large-scale economic synthesis of antisense phosphorothioate analogues of DNA for preclinical investigations. Ann. NY Acad. Sci. 616, 173–183.

    Article  CAS  Google Scholar 

  69. Zon, G. and Geiser, T. (1991) Phosphorothioate oligonucleotides: Chemistry, purification, analysis, scale-up and future directions. Anticancer Drug Design, 6, 539–568.

    CAS  Google Scholar 

  70. Lesnikowski, Z. J. and Jaworska, M. M. (1989) Studies on stereospecific formation of p-chiral internucleotide linkage. Synthesis of (R P,R P)-and (S P,S P)-thymidylyl (3′,5′)thymidylyl (3′,5′)thymidine di(O,O,-phosphorothioate using 2-nitro-benzyl group as a new S-protection. Tetrahedron Lett. 30, 3821–3824.

    Article  CAS  Google Scholar 

  71. Stec, W. J., Grajkowski, A., Koziolkiewicz, M., and Uznanski, B. (1991) Novel route to oligo(deoxyribonucleoside phosphorothioates. Stereocontrolled synthesis of P-chiral oligo(deoxyribonucleoside phosphorothioates) Nucl. Acids Res. 19, 5883–5888.

    Article  CAS  Google Scholar 

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  1. Lynx Therapeutics, Inc., Foster City, CA

    Gerald Zon

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  1. Gerald Zon
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  1. Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts

    Sudhir Agrawal

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© 1993 Humama Press Inc., Totowa, NJ

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Zon, G. (1993). Oligonucleoside Phosphorothioates. In: Agrawal, S. (eds) Protocols for Oligonucleotides and Analogs. Methods in Molecular Biology, vol 20. Humana Press. https://doi.org/10.1385/0-89603-281-7:165

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  • DOI: https://doi.org/10.1385/0-89603-281-7:165

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-281-1

  • Online ISBN: 978-1-59259-507-5

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